Transcriptional Regulation of Microglial Metabolic and Activation States by P2RY12

Microglia are the resident immune cells of the CNS. Under homeostatic conditions, Microglia play critical roles orchestrating synaptic pruning, debris clearance and dead cells removals. In disease, they are powerful mediators of neuroinflammation, as they rapidly respond to injury or infection within the CNS by altering their morphology, proliferating, and releasing cytokines and other signaling molecules. Understanding the molecular pathways involved in microglial function is pivotal for advancing neurobiological research and developing effective strategies for CNS disorders. In this context, P2RY12 is a G protein-coupled receptor (GPCR) that is uniquely enriched in microglia in the parenchyma and a canonical marker of homeostatic, ramified microglia. However, P2RY12 is downregulated in activated microglia and in neurological conditions. The consequences of P2RY12 downregulation in disease-associated microglia and how they influence microglial activation remain poorly understood. In this study, we apply transcriptional and histological methods to explore the changes to microglia upon a genetic P2RY12 loss. Our findings reveal that P2RY12-deficient microglia experience alterations in distinct metabolic pathways while preserving overall homeostatic microglial transcriptional identity. Lack of P2RY12 alters signature genes involved in homeostatic iron metabolism. Importantly, the genes encoding proteins in the Glutathione Peroxidase 4 (Gpx4)-Glutathione (GSH) antioxidant pathway related to ferroptosis susceptibility are impaired upon microglial activation with lipopolysaccharide (LPS) treatment. These results highlight the critical role of P2RY12 in regulating microglial immune and metabolic transcriptional responses under both homeostatic and inflammatory conditions, providing insights into its involvement in CNS pathophysiology. Proposed Model O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/665960v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@176a44corg.highwire.dtl.DTLVardef@1ca463org.highwire.dtl.DTLVardef@6c68dforg.highwire.dtl.DTLVardef@15913f9_HPS_FORMAT_FIGEXP M_FIG C_FIG At basal condition the transcriptional landscape of P2RY12-deficient microglia suggests that the cell is in a highly demanding metabolic state with increased oxidative phosphorylation and glycolysis as well as augmented expression of ribosomal proteins involved in cytoplasmic translation. Additionally, P2RY12-deficient microglia exhibit heightened expression of key regulators of the antioxidant response, suggesting elevated ROS levels as a consequence of this highly metabolically active and energetically demanding microglial state. Elevated ROS levels might diminish microglial antioxidant reserves thus rendering them more vulnerable to additional oxidative stress pushing the redox balance. When activated by LPS treatment, P2RY12-deficient microglia exhibit an impaired responsiveness of system xc and downstream enzymes involved in GSH mediated antioxidant response that together with LPS induced intracellular iron sequestration(Francois et al., 2014; S. Liu, Gao, & Zhou, 2022; McCarthy et al., 2018; Sfera et al., 2018; Shin et al., 2018; Urrutia et al., 2013) suggesting that P2RY12-deficient microglia will show increased susceptibility to ferroptosis. It is possible that system xc unresponsiveness in KO microglia results from the upregulation of ribosomal proteins involved in the inhibition of MD2, a negative regulator of p53 DNA binding activity(Daftuar, Zhu, Jacq, & Prives, 2013). In this sense, the repression of p53 inhibition will enhance its function, possibly explaining a suppression of Slc7a11 expression in LPS-activated P2RY12 deficient microglia.